Exoelectrogens Leading to Precise Reduction of Graphene Oxide by

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Exoelectrogens leading to precise reduction of graphene oxide by flexibly switching their environment during respiration Prerna Bansal, Sejal Doshi, Ajay Singh Panwar, and Dhirendra Bahadur ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b04390 • Publication Date (Web): 19 Aug 2015 Downloaded from http://pubs.acs.org on September 10, 2015

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ACS Applied Materials & Interfaces

Exoelectrogens Leading to Precise Reduction of Graphene Oxide by Flexibly Switching Their Environment during Respiration a Prerna Bansal , Sejal Doshi b, Ajay S. Panwar c and Dhirendra Bahadur*d

[a] Prerna Bansal Department of Metallurgical Engineering and Materials Science IIT Bombay, Mumbai, 400076 (India) E-Mail: [email protected] [b] Sejal Doshi Department of Metallurgical Engineering and Materials Science IIT Bombay, Mumbai, 400076 (India) E-Mail: [email protected] [c] Ajay S. Panwar Department of Metallurgical Engineering and Materials Science IIT Bombay, Mumbai, 400076 (India) E-Mail: [email protected] [d] Dhirendra Bahadur Department of Metallurgical Engineering and Materials Science IIT Bombay, Mumbai, 400076 (India) E-Mail: [email protected]

* Corresponding author: Fax: +91 22 2572 3480. E-mail address: [email protected] (D. Bahadur). ACS Paragon Plus Environment


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Abstract Reduced graphene oxide (RGO) has been prepared by a simple, cost effective and green route. In this work, graphene oxide (GO) has been reduced using gram negative facultative anaerobe S. dysenteriae having exogenic properties of electron transfer via electron shuttling. Apparently, different concentrations of GO were successfully reduced with almost complete mass recovery. Effective role of lipopolysaccharides has been observed while comparing RGO reduced by S. dysenteriae and S. aureus. It was observed that the absence of lipopolysaccharides in gram positive S. aureus leads to disrupted cell wall and S.aureus could not survive in the presence of GO, leading to poor and inefficient reduction of GO as shown in our results. However, S. dysenteriae having an outer lipopolysaccharide layer on its cell membrane reduced GO efficiently and the reduction process was extracellular for it. RGO prepared in our work has been characterized by X-Ray diffraction, Zeta potential, X-ray photoelectron spectroscopy and Raman spectroscopy techniques and the results were found to be in good agreement with that of chemically reduced GO. As agglomeration of RGO is major issue to overcome while chemically reducing GO, we observed that RGO prepared by bacterial route in our work has zeta potential value of -26.62mV, good enough to avoid restacking of RGO. Role of exoelectrogens in electron transfer in the extracellular space has been depicted. Toxin released extracellularly during the process pave tribute for reduction of GO due to its affinity towards oxygen.

Keywords: Graphene; biosynthesis; bacterial reduction; reduced graphene oxide; Shigella dysenteriae; mass production; green methodology.

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1. Introduction Graphene is an adept material with applications ranging from electronics to biosensors. thermal

3

This 2D sheet of carbon atoms has outstanding electronic 1, mechanical 2,

and optical properties

4

which make it useful in a wide spectrum of applications.

Mass production of graphene in a cost effective manner has been a major challenge for utilizing the full potential of this excellent material. For mass production of graphene, some alternative approaches need to be catered as the chemical synthesis route involves use of hazardous and carcinogenic chemicals

5-6

and high temperatures 7. Among many top down

and bottom up approaches to produce graphene and its derivatives

4-19

, graphite oxidation-

reduction has emerged as a better approach in terms of high yield and low cost process 6, 12-13, 17

. Photocatalytic 20 and electrochemical methods 21, UV radiation 22 and use of Vitamin C23,

suger24, glocose25, green tea26 and cross-linking polymers

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have also been reported for

reduction of graphene oxide (GO). Reduction of GO using bacteriorhodopsin under irradiation was also reported 28. Recently, micro-organisms

29-34

have been used as a green route for reduction of GO.

Such biological paths have shown some promising results for GO reduction. It has been reported that the Shewanella cells can reduce GO through an electron transfer process mediated by cytochromes MtrA-C/OmcA 29-31. In another study, GO sheets support growth of E.coli as biocompatible sites, while reduced graphene oxide (RGO) sheets were found to be growth inhibitor for E. coli 32-33. RGO nanowalls were found to be more toxic to the bacteria than the unreduced GO nanowalls

35

. Non-pathogenic extremophilic bacteria has also been

used for GO reduction 34. GO has been reported as an enhancer of cellular growth by helping cell proliferation 36. Trapping of bacteria by RGO and its inactivation has also been reported 37

. Interaction of GO with common human gut bacteria B. adolescentis indicated that GO

sheets promoted its proliferation, indicating good biocompatibility of GO 38.



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Such reports indicate that GO has been reduced using bacterial cells/biomass like Shewanella

29-31

and E.coli

32-33

. However there are clear gaps and many unsolved issues

associated with reduction of GO through bacterial route. Some of these issues include importance of bacterial cell membrane, least time consumption, maximum material yield, role of extracellular environment during interaction of bacterial cells with GO and cost effective mass production of high quality RGO, which have not been addressed clearly so far. Present work is an attempt to answer these concerns and to bridge the gaps for achieving GO reduction by green biological route using bacterial cells. Also, so far GO is reduced using anaerobic bacteria, hence this work solves a fruitful purpose of using aerobic environment to reduce GO. We have observed that gram negative Shigella dysenteriae has never been used for GO reduction till date. S. dysenteriae is a gram negative coccobacilli 39. Due to its facultative nature, it can flexibly change its respiratory environment from aerobic to anaerobic, thus having a tendency to survive for longer duration, which may be advantageous while using it for GO reduction. It was chosen as a model for GO reduction via electron shuttling and extracellular electron transport mechanism. S. dysenteriae when present in water causes severe dysentery, a pathogenic disease like shigellosis with shiga toxin 40 being the causative agent, but herein, shiga toxin shall be used for GO reduction, avoiding use of harsh chemicals. Shiga toxin present in the periplasmic space of the S. dysenteriae cells plays an important role in the electron transportation along with the cytochrome bd oxidase hooked on the outer membrane of the bacterial cell 41. Being a gram negative bacteria, S. dysenteriae has an advantage due to presence of lipo-polysaccharides layer in its outer cell membrane while gram positive bacterial cell wall like S. aureus lacks this layer. These capacities of S. dysenteriae motivated us to use it for the first time, as a novel model for studying the


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concerns related to biological approaches for reduction of GO. S. dysenteriae is known to be a human pathogen but the strain used in our work is of laboratory standards. Further, a comparative study was endeavored using gram negative S. dysentraie and gram positive S. aureus. We observed that lipopolysaccharide holds responsibility for survival of gram negative bacterial cells in the presence of GO. Our bacterially- reduced graphene oxide (Br-RGO) samples were analyzed by X-Ray diffraction (XRD), Zeta potential, X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy techniques and the results for Br-RGO samples were found to be in good agreement with the reported results for chemically synthesized RGO5-6, 13.

2. Materials and Methods 2.1 Synthesis of Graphite Oxide Improved Hummer’s method 12 was followed to oxidize pure graphite flakes (SigmaAldrich, 45µm, 99.99%). For this, a 9:1 mixture of concentrated H2SO4/H3PO4 (60:6.66 ml) was added to a mixture of graphite flakes (500mg, 1 wt equivalent) and KMnO4 (3.0 g, 6 wt equivalent), producing a slight exotherm to 45-55 °C. This reaction was again heated at 55 °C with continuous stirring for 12 h. The reaction mixture was then cooled to room temperature and kept in an ice bath with 30% H2O2. After cooling, the obtained solution was filtered using nylon filter paper (0.2µm pore size), using vacuum filtration assembly. The residue was washed with water, 30% HCl and ethanol alternately for several times. Later, the obtained material was coagulated with di-ethyl ether and filtered. It was then vacuum dried at 60 °C overnight, which gave approximately 1gram of graphite oxide powder. For the bacterial reduction of GO, graphite oxide aqueous suspension (0.1mg/ml) was prepared by sonicating graphite oxide powder (1mg) in 10 ml of sterile distil water for 1 h till a homogeneous brown



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dispersion was obtained. For mass reduction of GO, different concentrations (0.2mg/ml1.2mg/ml) were prepared using the similar protocol.

2.2 Preparation of bacterial inoculum and reduction of GO Bacterial strains of S. dysenteriae and S. aureus were procured from K. C. College, Mumbai, India. These cultures were initially inoculated from patients infected with the respective bacteria. They have been subcultured since last few years and hence assumed to have least virulence. Nutrient broth media was procured from Himedia. 2.2.1 Growth curve: S. dysenteriae and S. aureus were studied to obtain the mid-log phase. Mid-log phase refers to the period during which bacteria in culture are dividing rapidly and cell density of the culture increases many fold. It is that stage of log phase or exponential phase where cell division is at its peak, ready for any reaction. Briefly, 5 ml of overnight grown culture of S. dysenteriae and S. aureus were added to individual flat bottom conical flask containing 100 ml autoclaved nutrient broth and incubated at 37 °C in an orbital shaker at 200 rpm. Periodically OD was measured at 540 nm after every 5 minutes. 2.2.2 Reduction of GO: Overnight grown culture of S. dysenteriae and S. aureus were inoculated in 9 ml sterile nutrient broth in sterile glass bottle individually. Control was sterile nutrient media to check for sterility of media and S. dysenteriae and S. aureus inoculated in nutrient media individually. On acquiring the mid-log phase as calculated before, dispersed GO was added to the culture grown upto its exponential growth phase and incubated at 37 ºC on an orbital shaker at 200 rpm for 10 h. All experiments were performed in triplicates. Br-RGO was observed settled at the bottom of the glass bottle in nutrient media indicating reduction of GO. This solution was centrifuged at 5000 rpm for 1 min and the supernatant was decanted. Br-RGO so obtained was washed once with ethanol to remove any bacterial debris and centrifuged twice with water. The residue in the centrifuge tube was


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dried in vacuum oven overnight at 60 ºC giving Br-RGO powder which was further used for different characterizations.

2.3 Materials characterization Crystalline nature of GO and Br-RGO was measured by XRD using Philips powder diffractometer (PW3040/60) with Cu Kα (1.5406 Ǻ) radiation. Raman spectra was recorded using LabRAM HR 800 micro-Raman microscope (514.5 nm argon laser). FTIR spectra was obtained by JASCO spectrometer (6100 type-A). Zeta potential was measured by zeta potential analyzer (DelsaNano Corp.) to analyze the surface charges. Morphology of GO and Br-RGO was analyzed by high resolution transmission electron microscope (HRTEM) and field emission gun scanning electron microscopy (FEGSEM). Selected area electron diffraction (SAED) pattern was observed by HRTEM. X-ray photoelectron spectroscopy (XPS) measurements were done to analyze the molecular bonding of as-synthesized GO and Br-RGO using an electron spectroscopy chemical analysis probe (MULTILAB, Thermo VG Scientific) with a monochromatic Al Kα radiation of energy ~1486.6 eV.

3. Results and Discussion 3.1 Extracellular interaction and GO reduction by gram negative facultative anaerobe Shigella dysenteriae Here we present reduction of 0.1 mg/ml GO by gram negative facultative anaerobe S. dysentriae. GO was added during the mid log phase and its spontaneous conversion to BrRGO was observed. S. dysenteriae in mid-log phase was achieved after 3 h on addition of overnight grown inoculum. Reaction mixture was incubated in orbital shaker for 10 h at 200 rpm at 37 °C. Further, different concentrations of GO (0.2 mg/ml to 1.2 mg/ml) were added to S. dysenteriae and incubated at 37 °C in orbital shaker for 10 h at 200 rpm. Figure 1 shows



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different concentrations (0.2mg/ml to 1.2mg/ml) of Br-RGO reduced in presence of S.dysentraie.

Figure 1 As-synthesized Br-RGO for different GO concentrations (0.2- 1.2mg/ml) in presence of S. dysentriae (Sd). A visible color change of as-synthesized GO (brown) to Br-RGO (black) was observed settled at the bottom of the bottle in nutrient media. This color change from brown to black indicated the reduction of GO to Br-RGO. On the other hand, there was no visible color change seen in control experiments (Figure S1 in supporting information) with nutrient broth media alone without any bacterial species. Br-RGO was recovered successfully after removal of the bacterial debris by multiple washing with ethanol and water. It was used for further characterization to ensure the quality of as-obtained Br-RGO. Addition of GO to the grown culture during mid-log phase holds significance as exponential growth phase is the most active phase of the bacterial growth cycle. At this stage cell mass and cell number in the cultured media increase exponentially with time and cell population doubles at regular intervals of time. Growth of the cells depends on the presence of ambient energy source in the form of nutrients, salts and presence of dissolved oxygen in the system. In addition, this energy is used to produce new cells and for the maintenance of existing cells. In our work, other than the media components, GO also acts as an oxygen source for cellular growth and that way GO itself gets reduced. Also, we observed the 8 ACS Paragon Plus Environment

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simultaneous formation of thin layers of Br-RGO on the surface of the media at air water interface during the experiment. Due to aerobic environment, bacteria at the surface of the media also hold capacity to reduce GO as the bacteria present in the media. Hence, S. dysentriae is an apt model for GO reduction in extracellular environment.

3.1.1 Raman Spectra of Br-RGO reduced by S.dysentraie (Sd) Confocal Raman measurements were taken using WiTec Alpha300 Confocal Raman microscope with 532 nm wavelength incident laser light using 50× objective lens. For all the bacteria samples, measurements were taken at 5mW incident laser power to avoid any damage to the sample caused by laser induced heating.

Figure 2 Raman spectra of (a) Br-RGO after removal of Sd cells, (b) Br-RGO in media before removal of Sd cells, (c) Sd cells in media, and (d) Graphite Oxide.



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Raman spectroscopy is a well known technique to characterize crystal structure, disorder and defects in graphene and related materials. One can determine the degree of reduction of GO by Raman spectra by the changes in relative intensity of two prominent ‘D’ and ‘G’ peaks 42-43. It is well known that Raman spectra of pure graphite shows a strong ‘G’ peak at ~1570 cm−1 due to the first order scattering of E2g phonon of sp2 carbon atoms43. A broadened ‘D’ peak appears in the Raman spectra of graphite oxide at ~1350 cm−1, due to the reduction in size of in-plane sp2 domains of graphite induced by the creation of defects and distortions of the sp2 domains. It is common to all sp2 carbon lattices and arises from the stretching of C-C bond. Figure 2 shows the Raman spectra of graphite oxide with ‘D’ peak at 1349 cm-1 and ‘G’ peak at 1581 cm-1. The relative intensity ratio of both peaks (ID/IG) is a measure of disorder degree and is inversely proportional to the average size of the sp2 clusters. The ratio of the ‘D’ peak intensity to the ‘G’ peak intensity (ID/IG) for graphite oxide is ~0.84. ID/IG ratio for Br-RGO (reduced using Sd) is ~1.15. Such an increase in ID/IG for BrRGO is in good agreement with the reported values of ID/IG for chemically synthesized RGO6. It suggests that more graphitic domains were formed and the sp2 cluster number is increased post reduction, indicating good reduction efficiency of Shigella5 6. Raman spectra for Br-RGO in media before removal of S. dysenteriae (Sd) shows ‘D’ and ‘G’ peaks similar to Br-RGO with an extra peak at ~2930 cm-1. A similar peak is seen in the Raman spectra of Sd corresponding to CH3 symmetric stretch band near 2935 cm−1 (‘B’ peak), indicating presence of phospholipids which is a major constituent of cell membrane 44. A decrease is observed in the ID/IG ratio of Br-RGO after removal of the bacteria with ethanol, which might be due to reaction of ethanol with the residual oxygen functional groups of Br-RGO.


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3.1.2 Zeta potential measurements of Br-RGO reduced by S.dysentraie Surface charge (zeta potential) values for (a) S. dysenteriae cells in media (b) Br-RGO before removal of Sd cells in media (c) Br-RGO after removal of Sd cells and (d) Graphite Oxide aqueous suspension at pH 6.8 ± 0.2 are presented in Figure 3.

Figure 3 Zeta potential values for (a) S. dysenteriae cells in media (b) Br-RGO before removal of Sd cells in media (c) Br-RGO aqueous suspension after removal of Sd cells and (d) Graphite Oxide aqueous suspension Zeta potential value for S. dysenteriae is -19.16mV, which indicates that bacteria surface are negatively charged. We observed that aqueous suspension of graphite oxide is highly negatively charged (-38.18mV) which is due to the ionization of the carboxylic acid and hydroxyl groups which are present on edges and basal plane of GO sheets 5. Zeta potential value for Br-RGO without removal of bacterial debris was found to be (-20.47mV), slightly more negative than Sd. We observed that Zeta potential value for Br-RGO increased after removal of bacterial debris with ethanol and water. Zeta potential value for Br-RGO (26.62mV) is lower than GO (-38.18mV) due to removal of oxygen functional groups after reduction by Sd cells. All the zeta potential values for graphite oxide, Br-RGO and bacteria 11 ACS Paragon Plus Environment


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were in good agreement with their reported values for chemically synthesized RGO 6. As agglomeration of RGO is a major issue to overcome while chemically reducing GO

27

, we

observed that Br-RGO prepared by bacterial route in our work has zeta potential value (26.62mV) good enough to prevent restacking of Br-RGO.

3.1.3 FTIR measurement of Br-RGO reduced by S.dysentraie Many distinct vibrational modes of various oxygen functionalities were observed in the Fourier-transform infrared (FTIR) spectra of graphite oxide and Br-RGO presented in Figure 4, recorded using KBr as reference. FTIR spectra of graphite oxide shows a sharp broad peak of –OH stretching at nearly 3400 cm-1 and peak corresponding to bound H2O molecules is observed at ~1363cm-1.

Figure 4 FTIR spectra of Graphite Oxide and Br-RGO reduced by Sd cells.


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FTIR spectra of graphite oxide indicated graphite oxidation to great extent as confirmed by the presence of peaks corresponding to various oxygen functionalities like – C=O (stretching of carbonyl and carboxyl groups at GO edges), C-O and >O (epoxide) groups present at ~1730, ~1250 and ~1038 cm-1 respectively. In-plane C=C bonds were seen to be present at ~1615 cm-1. FTIR spectra of Br-RGO confirmed the efficient reduction of GO in presence of Sd, indicated by a significant decrease in the intensity of peaks corresponding to various oxygen functionalities due to removal of partial carboxyl, hydroxyl and epoxide groups from GO and also due to cracking of aromatic C=C bonds 6, validating the reduction of functional groups by Sd cells. Also, C=C(aromatic stretch), and C-O were slightly shifted due to the change of hydrophobic environment present in the reduced sample. Our FTIR result was found in good agreement with chemically synthesized RGO16.

3.1.4 HRTEM Results: Extracellular GO reduction by S. dysenteriae Gram negative bacteria (S. dysenteriae) has lipopolysaccharide in outer part of its cell membrane whereas it is absent in gram positive bacteria (S. aureus). Figure 5a presents HRTEM micrograph of control S. dysenteriae (Sd) cells in media, indicating intact cell membrane. Figure 5b shows HRTEM image of Sd after interaction with GO. It depicts that GO reduction by gram negative Sd is an extracellular process, as Br-RGO was found lying in the vicinity of the bacterial solution and the cell wall of the bacteria was intact. Various oxygen functional groups present in GO acted as a source of oxygen for the growth and maintainence of Sd cells. No damage was caused to Sd cells by GO. Hence, it was assumed that lipopolysaccharides may probably have a key role in extracellular interaction between gram negative Sd cells and GO. TEM results (Fig. S2(b) in supporting informarion) indicated the similar extracellular interaction between GO and Sd. Selected area electron diffraction



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(SAED) pattern of Br-RGO (Figure 5d) showed hexagonally packed lattice whereas no diffraction spots or rings were seen for Sd bacteria as expected.

Figure 5 HRTEM images of (a) Sd cells (b) Sd cells with Br-RGO sheets showing extracellular interaction (c) Br-RGO sheets after removal of bacterial debris (d) Diffraction patterns for Br-RGO and Sd bacteria cells, recorded for two different areas marked with an arrow.

3.1.5 XPS Analysis of GO and Br-RGO reduced by S.dysentraie To understand the extent of GO reduction through bacterial route, XPS analysis was performed for GO and Br-RGO samples. To carry out XPS measurements samples were drop casted on a Si substrate. Figure 6(a) presents C1s spectra of GO, which is Gaussian fitted (using software XPS Peak 4.1 with shirley background substraction) as a superposition of four peaks at 284.8, 287.3, 289.6 and 282eV. These peaks correspond to non-oxygenated ring


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carbon (C-C), carbonyl carbon (C=O), carboxylate carbon (O=C-OH) and C-Si respectively (due to Si substrate used for XPS measurements), indicating a considerable degree of oxidation of GO.

Figure 6 C1s XPS spectra of (a) GO and (b) Br-RGO Figure 6(b) presents C1s XPS spectra for Br-RGO, indicating a significant reduction in the O/C fraction from ~2.4 for GO to ~0.42 for Br-RGO, due to decrease in the peak intensity of oxygen functional groups of GO. It shows efficient reduction of GO in presence of Sd. For the hydrazine reduced GO, O/C fraction is reported to vary from 0.21 to 0.09 15 ACS Paragon Plus Environment


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depending upon the amount of hydrazine added and time of contact with hydrazine45. We observed that post reduction peak intensities for carbonyl carbon (C=O) at 287.3 eV and for carboxylate carbon (O=C-OH) at 289.6 eV were decreased to a great extent indicating considerable deoxidation of GO by Sd. An increase in the intensity of peak at 284.6 eV post reduction indicated the restoration of sp2 bonded carbon after reduction46. The analysis of the peak area ratio of four components of C1s spectra of GO confirmed that ~30% of carbon in GO is not bound to any oxygen functionality while for Br-RGO the same is found to be ~70%. Such a decrease in the intensity of oxygen functionalities present in GO after interacting it with Sd is in good agreement with chemically synthesized RGO45-47. Our XPS results for GO and Br-RGO are in well accordance with our FTIR results indicating decrease in O/C fraction upon reduction.

XRD results (Fig. S3 in supporting information) indicated a shift in the position of [002] diffraction peak of GO from 2θ ~9.56° to 2θ ~23°, indicating reduction of GO to BrRGO by Shigella cells 6.Also, broadening of [002] peak upon reduction indicated a decrease in crystalline nature due to insertion of defects and distortions of sp2 lattice upon reduction. These results are well in agreement with our Raman studies which indicated an increase in ID/IG value for Br-RGO, leading to more defects. UV–vis absorption studies (Fig. S4 in supporting Information) of pure GO and Br-RGO showed that upon reduction there is a significant red shift in the position of peak at 230 nm for Br-RGO sample in the absorption spectra which indicated the restoration of electronic conjugation after reduction by Shigella cells 16.

3.1.6 Possible mechanism for GO reduction by gram negative S.dysentraie


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Facultative anaerobes, especially Shigella spps. have a tendency to switch respiration cycle as the environment facilitates. Most likely they use the aerobic way of respiration, thus releasing 2 molecules of ATP. Nitrate reductase generates proton motive force and ATP synthase bound to the membrane serves physiological function of transforming electrochemical potential across the membrane, synthesizing ATP from ADP with the help of electrochemical gradient. Growth of new cells takes place actively during the exponential growth phase; cells require more and more energy from the available sources in their environment.

Figure 7 Possible mechanism for GO reduction in presence of Sd

For this energy generation cells consume oxygen from GO and hence GO itself gets reduced. On the other hand, oxygen supply borrowed from GO plays a role in electron transport chain, which oxidizes NADH and FADH2 back to NAD+ and FAD+, which are involved in the four reduction reactions of the respiration cycle. In S. dysenteriae NADH is



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reduced to NAD+ in the presence of NADH reductase. The extracellular interaction between S. dysentriae and GO in present work has been attributed to periplasmic toxin Shiga as it has various receptor sites like glycoconjugates that includes oxygen48. In addition, cytochrome bd oxidase and aa3 oxidase equally contributes for electron transportation (as shown pictorially in Figure 7). The efficient energy of aa3-type cytochrome c oxidase supercomplex yields the enzyme having greater affinity for oxygen.

3.2 GO interaction with gram positive S. aureus It is well known that gram negative bacteria has lipopolysaccharide in outer part of its cell membrane whereas it is absent in gram positive bacteria. As seen in our results for gram negative Sd, cell membrane was found intact after interaction with GO, indicating extracellular interaction between GO and gram negative Sd. To understand the role of bacterial cell membrane, we have conducted comparative study between gram negative and gram positive bacteria. In our work we have selected S. aureus as model for gram positive cocci. While choosing the two model bacteria for our work, we took enough care to select species which belong to different genus; S in S. dysentraie stands for Shigella whereas in S. aureus it stands for Staphylococcus. This section presents our results related to GO interactions with gram positive S.aureus.

3.2.1 FEGSEM Results: Interaction between GO and S. aureus Figure 8 presents FEGSEM micrograph of control image of S.aureus cells showing intact cell membrane before its interaction with GO.


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Figure 8 (a) and (b) FEGSEM image of S.aureus cells before interaction with GO (Images were taken at different magnifications).

Figure 9 (a) and (b) FEGSEM images of gram positive bacteria S.aureus after interaction with GO (Images were taken at different magnifications).

From our FEGSEM micrographs, presented in Figure 9 (a,b), we observed disrupted cell wall with spikes all around S.aureus after its interaction with GO, while for control S.aureus cells, presented in Figure 8 (a,b), bacterial cell membrane was found intact before interaction with GO. Such cell wall rupture seen for S.aureus cells after interaction with GO 19 ACS Paragon Plus Environment


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did not happen in our work with Sd cells (section 3.1.4). We found that during reduction of GO, outer surface of S.aureus was completely denatured leading to cell death. This activity had occurred in S.aureus due to the absence of lipopolysaccharide layer unlike in gram negative bacteria Sd. Lipopolysaccharide (LPS) is a lipid bilayer present in the outermost part of gram negative bacterial cell membranes whereas it is absent in gram positive bacteria. It consists of lipids and polysaccharides joined together covalently. LPS mainly involves O antigen, core oligosaccharide and lipid A

49

. Lipid A layer is well known to act as endotoxin, thus

responsible for the structural integrity and toxicity of gram-negative bacteria. This layer makes the cell membrane more negatively charged which leads to more stabilized membrane structure protecting the gram negative cell membrane from specific chemicals. Due to an outer protecting lipopolysacharride layer, gram negative Sd survives in the presence of GO and leads to efficient GO reduction while the absence of lipopolyscharride layer in gram positive Sa leads to cell death as confirmed by our morphological studies. Another point of notice was the growth rate of Sa which was slower as compared to Sd. Thus the time of interaction with GO for reduction was lower in case of Sa. Due to deficiency of interaction between oxygen molecule from GO and molecular signal in the form of enzyme, distinguished event has occurred in Sa rendering it to be a weak tool for GO reduction. Further, we observed that the bacterial morphology either rod shape (Sd) or spherical shape (Sa) does not play any role for GO reduction as whatever be the shape, both the bacterial surfaces were negatively charged. Zeta potential values for Sd and Sa cells in media were found to be -19.16mV and -18.95mV, respectively. It indicates that Surface potentials were almost same (-19±0.5) for both the bacterial morphologies.


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4. Conclusions Efforts to produce environmentally benign method for reduction of GO have been achieved successfully. Thereby, bacterial reduction of GO sturdily rely on the structure of bacterial cell membrane, in particular, lipopolyscharrides layer beneath the outer membrane. Due to availability of lipopolyscharrides layer under the outer membrane of gram negative bacteria (S.dysentraie), GO is reduced extracellularly. As lipopolysaccharide layer is absent in gram positive bacteria (S.aureus), hence, it could not survive in the presence of GO and reduction of GO was not achieved by it. Apparently bacterial morphology had no role on the reduction of GO through bacterial route. It was observed that, if GO was added during midlog phase of bacterial growth cycle, then Br-RGO formation was instant. Present investigation for GO reduction using S.dysentraie is a low cost, effective, easy, reproducible, and energy efficient method. FTIR spectra of Br- RGO confirmed the efficient reduction of GO in presence of S. dysenteriae, indicated by a significant decrease in the intensity of peaks corresponding to various oxygen functionalities due to removal of partial carboxyl, hydroxyl and epoxide groups. XPS measurements performed on Br-RGO showed a significant reduction in the O/C fraction due to decrease in the peak intensity of oxygen functional groups, indicating efficient reduction of GO in presence of S.dysentraie. Zeta potential value for Br-RGO (-26.62mV) is lower than GO (-38.18mV) due to removal of oxygen functional groups after reduction by S. dysentraie cells. Above results lead to a path that microbe with redox potential has a promising impendence to reduce GO. It is now, convincing to use bacteria available in the environment that cause nuisance in the form of dreadful diseases, for a good cause like processing graphitic materials. With this study it is shown that lipopolysacharride along with exoelectrogens are responsible for reducing GO. Avoiding use of hazardous chemicals,



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whereas imbibing green route to synthesize nanomaterials for mass production with consistent reproducibility was the target achieved.

Acknowledgement Authors would like to thank Nanomission-DST, Government of India for providing financial support to conduct this research. Also they greatly acknowledge the central characterization facilities at SAIF, IIT Bombay, India.

Supporting Information Control experiment: GO interaction with nutrient broth media (without any bacterial species), TEM Results: Extracellular interaction between GO and S. dysenteriae cells, XRD Spectra of Br-RGO, Optical Studies of bacterially reduced graphene oxide samples. This material is available free of charge via the Internet at http://pubs.acs.org.

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Table Of Contents (TOC) graphic: Schematic representation of bacterial reduction of graphene oxide: A comparative study between gram negative (S. dysenteriae) and gram positive (S. aureus) bacteria.

Extracellular Reduction

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